Abstract 18160: Distinct Transcriptomic Signatures Across Postnatal Cardiomyocyte Development
Background: The murine heart undergoes profound growth between postnatal day 10 (P10) and P18 due to both cardiomyocyte (CM) hypertrophy and hyperplasia.
Objective: To determine the dynamic changes in the cardiomyocyte (CM) transcriptome, including mRNAs, lncRNAs and miRNAs, during this critical period, which have not been characterised previously.
Methods: We developed a standardised procedure for the rapid isolation of highly pure (~95%) and viable (~80% of cells) CMs from neonatal (P1) through to adult hearts by Langendorff retrograde perfusion and immunomagnetic cell separation. Total RNA was extracted from CMs pooled from a litter of 6-8 mice at P2, P10, P13 and P70 (n=3), to generate poly(A) and small RNA libraries for Ion Torrent sequencing.
Results: Principal component analyses of mRNAs, lncRNAs and miRNAs revealed clustering of biological replicates into developmental time points, indicating high reproducibility. Over 3,000 differentially expressed (DE)-mRNAs and 231 DE-lncRNAs were identified between P2 and P10, P10 and P13, P13 and P70 (limma/VOOM; adj. p-value<0.05, fold change>1.5), with 226 DE-mRNAs between P10 and P13. CM-specific Gene Ontology term analyses confirmed DE-mRNAs upregulated at P10 compared to P2 are involved in aerobic respiration, muscle phenotype and fatty acid metabolism (p<0.01 Fisher’s exact test); moreover, consistent with adult CMs being terminally differentiated, downregulated genes at P70 compared to P13 are involved in DNA replication, cell cycle activity, mitosis and cell division. A number of functionally relevant mRNAs, lncRNAs and miRNAs showed concordant expression. Interestingly, the DE-lncRNA, H19, encoding miR-675, involved in skeletal differentiation and regeneration, was highly abundant in CMs at P2 but expression of both fell at later time points coincidently with the adjacent DE-mRNA, Igf2.
Conclusions: Our standardised CM purification procedure generates highly reproducible RNA-seq datasets that reveal distinct transcriptomic profiles along the postnatal developmental trajectory. They also highlight the coordinated expression patterns of mRNAs, lncRNAs and miRNAs during CM maturation that will be important for understanding underlying gene regulatory networks.
Author Disclosures: A.M. Nicks: None. S.R. Holman: None. A. Chan: None. D.T. Humphreys: None. N.J. Smith: None. J.W. Ho: None. N. Naqvi: None. A. Husain: None. R.M. Graham: None. S.E. Iismaa: None.
- © 2016 by American Heart Association, Inc.